The present invention concerns a process for the amplification of nucleic acid and a kit for the amplification of nucleic acid.
A prerequisite for the analysis, particularly the sequence analysis, of nucleic acids is that the nucleic acid to be analysed is available in adequate quantities. Particularly with nucleic acids in very low concentrations, their sequence analysis presents a technical problem. The state of the technology knows many processes for amplifying scarce nucleic acids, i.e. those occurring in low concentration, before the analysis.
In particular, the analysis of the 5xe2x80x2-terminal coding region of RNA molecules causes difficulties. A very recent, widespread technique for the amplification of nucleic acids is based on the polymerase chain reaction (the so-called PCR technique) for the in vitro amplification of DNA (see Sambrook et al, Molecular Cloning, Laboratory Manual, 2nd Edition, Chapter 14).
If the structure of RNA is to be elucidated, then before the actual amplification reaction a cDNA has to be prepared from the RNA to be analysed. In this, the problem often arises that the 5xe2x80x2 end of the RNA is transcribed into cDNA with low frequency, and hence a sequence analysis is more difficult to obtain than [for] more 3xe2x80x2-located RNA sections.
The state of the technology describes several possibilities for amplifying the 5xe2x80x2 end of RNA with the aid of the PCR technique. Common to most of these processes is that the first cDNA strand prepared from the RNA is extended at its 3xe2x80x2 end by addition of further nucleotides, so-called xe2x80x9ctailingxe2x80x9d, before the single-stranded cDNA strand is complemented to give the double-stranded cDNA.
As one possibility for the 3xe2x80x2 tailing of DNA, the attachment of an oligonucleotide fragment to the free 3xe2x80x2 end of cDNA or the polymerisation of desoxyribonucleotides onto the free 3xe2x80x2 end has been described.
However, these known tailing processes have the disadvantage that the reaction is difficult to control, the yields of 3xe2x80x2-extended molecules sometimes do not satisfy the requirements, and undesirable by-products arise, in that for example several oligonucleotides become ligated to one another, and thus the 3xe2x80x2 ends of different DNA molecules are not extended equally.
The technical problem on which the present invention was based was that of providing a further process for the amplification of a nucleic acid, with which the disadvantages arising from the state of the technology are not observed, and which in particular guarantees high efficiency in the 3xe2x80x2 extension of nucleic acids.
The above problem is solved according to the invention by a process wherein the tailing of the nucleic acid is effected by reacting the nucleic acid to be amplified with a ribonucleotide in the presence of terminal transferase (also called terminal desoxynucleotidyl-transferase (TdT)).
This process, also referred to below as CRTC (controlled ribonucleotide tailing of cDNA), uses the extremely effective and well controllable RNA polymerising activity of terminal transferase under normal tailing conditions. The normal tailing conditions are for example described in Deng G R and Wu R (1983), Methods in Enzymology, 100: 96-116 and Roychoudkurg R et al. (1976), Nucleic Acids Res., 3: 863-877. This TdT activity adds a limited number of ribonucleotides, which are used as triphosphates (rNTPs) to the 3xe2x80x2 end of a single-stranded or double-stranded nucleic acid, preferably a DNA. 2-4 ribonucleotides are preferably polymerised on.
In contrast to the primer-dependent DNA polymerase activity of TdT, which catalyses the repeated incorporation of desoxyribonucleotides (dNTPs) onto the 3xe2x80x2 terminus of a single- or double-stranded DNA, desoxyribonucleotide tailing, the ribonucleotide tailing of a single-stranded DNA is influenced neither by the nature of the bases attached (rGTP, rATP, rUTP or rCTP) nor by the nature of the 3xe2x80x2-terminal nucleotide of the DNA primer (GOH, AOH, TOH or COH). Under standard conditions, an efficiency of more than 95% is achieved with the ribo-tailing of cDNAs according to the invention. rGTP is quite especially preferred for the ribo-tailing.
The creation of a short homopolymeric attachment made of ribonucleotides at the 3xe2x80x2 end of the nucleic acid allows the unidirectional ligation of this nucleic acid, for example of a first cDNA strand, to a linearised plasmid DNA with a complementary 3xe2x80x2 overhanging end in the presence of a ligase, preferably T4 DNA ligase. Hence the process according to the invention renders superfluous the attachment of a section of homopolymeric desoxyribonucleotides, which is often difficult to optimise and to control and commonly leads to nonspecific PCR products, and the process according to the invention also avoids the problems which arise in the ligation of a single-stranded oligonucleotide (also called an anchor) to the 3xe2x80x2 end of cDNAs using T4 RNA ligase.
In the process according to the invention, a single-stranded DNA, and quite particularly a cDNA, is preferably used, this being preferably derived from a poly-(A)+-RNA.
In a preferred embodiment of the process according to the invention, after the tailing step a further nucleic acid molecule is bound to the 3xe2x80x2-end of the extended nucleic acid molecule.
The further nucleic acid molecule is preferably a double-stranded DNA and possesses a 3xe2x80x2 overhanging end, which is complementary to the 3xe2x80x2 end of the nucleic acid molecule extended by the ribonucleotides. This complementarity between the further nucleic acid molecule and the extended nucleic acid molecule favours the desired attachment of the further nucleic acid molecule through its hybridisation to the 3xe2x80x2 end of the extended nucleic acid molecule. This procedure is also described as xe2x80x9ctaggingxe2x80x9d.
In an especially preferred embodiment, the further nucleic acid molecule is a DNA vector or an adapter molecule (also called a linker), which preferably at the same time contains a recognition sequence for at least one restriction enzyme.
Further, it is preferred that the further nucleic acid molecule is not only hybridised, but also ligated, to the 3xe2x80x2 end of the extended nucleic acid molecule, preferably with T4 DNA ligase.
In a particularly advantageous embodiment, the further nucleic acid molecule is at the same time the primer for the synthesis of a second DNA strand, which complements the first DNA strand. The synthesis of the complementary strand can take place before the actual PCR amplification step, so that firstly a double-stranded nucleic acid is obtained, which is then amplified with the conventional PCR technique. However, the synthesis of the complementary strand (second strand synthesis) can take place at the same time as the PCR amplification.
The process according to the invention is suitable for all processes wherein DNA adapters are used and/or dNTP tailing takes place by means of TdT (such as for example in processes using the PCR-Select-cDNA-Subtraction Kit of Clontech, published in CLONTECHniques, October 1995), but it is especially suitable for the sequence analysis of RNA, in particular for the detection of the 5xe2x80x2 sequence of a sparsely occurring RNA.
Here it is firstly necessary that a first cDNA strand be prepared from the RNA to be analysed. This can be effected by processes known in the state of the technology. The single-stranded cDNA strand obtained is then amplified by the measures explained in detail above. The amplified double-stranded DNA obtained can next be subjected to conventional processes for the sequencing of DNA.
Furthermore, the present invention allows the provision of a kit for the amplification of any nucleic acid, the kit containing at least one ribonucleotide, preferably selected from rGTP, rATP, rUTP and rCTP, and the terminal transferase.